Potent and selective MAO-B inhibitory activity: Amino- versus nitro-3-arylcoumarin derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.12.001

In this study we synthesized and evaluated a new series of amino and nitro 3-arylcoumarins as hMAO-A and hMAO-B inhibitors. Compounds 2, 3, 5 and 6 presented a better activity and selectivity profile against the hMAO-B isoform (IC₅₀ values between 2 and 6 nM) than selegiline. In general, the amino derivatives (4-6) proved to be more selective against MAO-B than the nitro derivatives (1-3). Additionally, a theoretical study of some physicochemical properties, PAMPA and reversibility assays for the most potent derivative, and molecular docking simulations were carried out to further explain the pharmacological results, and to identify the hypothetical binding mode for the compounds inside the hMAO-B.

Full text of DOI:10.1016/j.bmcl.2014.12.001

Bioorganic & Medicinal Chemistry Letters 25 (2015) 642–648
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent and selective MAO-B inhibitory activity: Amino- versus
nitro-3-arylcoumarin derivatives
b
Maria João Matos a, , Fernanda Rodríguez-Enríquez , Santiago Vilar a,c, Lourdes Santana a, Eugenio Uriarte a,
⇑
George Hripcsak c, Martín Estrada d, María Isabel Rodríguez-Franco d, Dolores Viña b
a Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
b Departamento de Farmacología, CIMUS, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
c Department of Biomedical Informatics, Columbia University Medical Center, 10032 New York, USA
d Instituto de Química Médica, Consejo Superior de Investigaciones Científicas (IQM-CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study we synthesized and evaluated a new series of amino and nitro 3-arylcoumarins as hMAO-A
and hMAO-B inhibitors. Compounds 2, 3, 5 and 6 presented a better activity and selectivity profile against
the hMAO-B isoform (IC50 values between 2 and 6 nM) than selegiline. In general, the amino derivatives
(4–6) proved to be more selective against MAO-B than the nitro derivatives (1–3). Additionally, a theo-
retical study of some physicochemical properties, PAMPA and reversibility assays for the most potent
derivative, and molecular docking simulations were carried out to further explain the pharmacological
results, and to identify the hypothetical binding mode for the compounds inside the hMAO-B.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 24 October 2014
Revised 1 December 2014
Accepted 1 December 2014
Available online 8 December 2014
Keywords:
3-Arylcoumarins
Perkin reaction
Monoamine oxidase inhibitors
PAMPA assay
ADME theoretical properties
Docking studies
Neurodegeneration is a large class of chronic and progressive
nervous system biochemical processes based on constitutional
neuronal degeneration. The wrong regulation of these processes
leads to different neurodegenerative diseases (ND), such as
Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic
lateral sclerosis, and Huntington disease.1 Among them, AD and
PD are the most prevalent. The etiology of these diseases is not
completely known, but several risk factors have been identified,
including environmental factors, genetics, and age.1,2 Several
research hypotheses exist regarding the pathogenesis, including
different mechanisms that may be involved in the development
of the disease, such as oxidative stress, inflammation, excitotoxic-
ity, mitochondrial dysfunction and proteolytic stress.2 These
pathologies severely influence the life quality of the elderly.3
Therefore, studies in the area are continuously improving.
neurotransmitter, which showed low levels in the basal ganglia
in animal models of Parkinsonism. After the success of a placebo-
controlled trial, levodopa (LD) became an essential drug for the
treatment of PD.5 Even today the standard treatment of PD is the
combination of LD with inhibitors of peripheral dopa decarboxyl-
ase (DDC), to avoid the formation of DA in the periphery.5 LD is also
associated to inhibitors of the enzyme catechol-O-methyltransfer-
ase (COMT) and prevents the formation of 3-O-methyldopa
(3-OMD).6 Among other drugs that can be used to treat PD, selec-
tive inhibitors of monoamine oxidase B (MAO-B) play an important
role.7,8 MAO-B metabolizes approximately 80% of the DA in the
SNC, without affecting the serotonin or noradrenaline metabolism.
MAO is a flavin-containing enzyme bound to the mitochondrial
outer membrane of neuronal, glial, and other cells, which is
responsible for the oxidative deamination of endogenous mono-
amine neurotransmitters, trace amines, and a number of amine
xenobiotics.9 This enzyme exists in two distinct enzymatic iso-
forms, namely MAO-A and MAO-B encoded by different genes
and distributed in diverse tissues.10 Increased activity of MAO-A
and MAO-B is associated with loss of endogenous and exogenous
monoamine amounts.
PD, the second most important ND, is characterized by loss of
dopaminergic neurons in the nigrostriatal pathway. These neurons
extend from the zona compacta of the substantia nigra (SNC) to the
striatum.3 The symptoms of PD, resting tremor, rigidity and brady-
kinesia, appear when about 80% of dopaminergic neurons have
been destroyed.4 In the 50s, dopamine (DA) was described as a
An increase in MAO-B activity is also associated with gliosis,
which can result in high levels of hydrogen peroxide and oxidative
free radicals. Evidence exists that as aging progresses activity of
⇑
Corresponding author. Tel.: +34 881 814936; fax: +34 981 544912.
E-mail address: mariacmatos@gmail.com (M.J. Matos).
http://dx.doi.org/10.1016/j.bmcl.2014.12.001
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

M. J. Matos et al. / Bioorg. Med. Chem. Lett. 25 (2015) 642–648
643
MAO-B in the brain increases, but not so for MAO-A.8 The largest
increases are in the basal ganglia and thalamus, followed by the
frontal cortex and, to a lesser extent, cerebellum and parietal and
temporal cortices.11
Some studies have demonstrated the preliminary dominance of
coumarin compounds in the treatment of AD and PD for improving
the elderly’s life quality. Coumarins represent an important family
of naturally occurring12 and/or synthetic oxygen-containing het-
erocycles, bearing a typical benzopyrone framework.13 Therefore,
coumarinic compounds are a class of lactones structurally con-
and selectivity to MAO-B, also those derivatives with different het-
erocyclic rings in this position possess potency and selectivity
towards MAO-B.39
Therefore, almost all the studied derivatives might be consid-
ered as potential lead compounds for treating PD. Taking into
account this previous results, the introduction of nitro and amino
groups in the 3-aryl ring of 3-arylcoumarins seems to be of high
interest. A comparison between both substituents in different posi-
tions was performed in this new study. Docking calculations were
an important tool to support and better understand the results, and
also to the rational design of new derivatives.
structed by a benzene ring fused
a-pyrone ring, and essentially
possess conjugated system with rich electron and good
p
–
p
6-Methyl-3-nitrophenylcoumarins 1–3 were synthesized
starting from the commercially available 5-methylsalicylaldehyde
and the respectively substituted 3-nitrophenylacetic acids by a
modified Perkin reaction, in good yields (70–80%). The amino-
derivatives 4–6 were obtained, in very good yield (92–97%), from
1 to 3, respectively, in a palladium/carbon catalyzed reaction
(Scheme 1).40–42
The biological evaluation of the test drugs on hMAO activity
was investigated by measuring their effects on the production of
H2O2 from p-tyramine (a common substrate for hMAO-A and
hMAO-B), using the AmplexÒ Red MAO assay kit and microsomal
MAO isoforms prepared from insect cells (BTI-TN-5B1-4) infected
with recombinant baculovirus containing cDNA inserts for
hMAO-A or hMAO-B.43,44 New compounds and reference inhibitors
were unable to react directly with the AmplexÒ Red reagent, which
indicates that these drugs do not interfere with the measurements.
On the other hand, in the experiments and under the experimental
conditions, hMAO-A displayed a Michaelis constant (Km) equal to
charge-transport properties.14 The simplicity and versatility of
the coumarin scaffold make it an interesting starting-point for a
wide range of biological applications.15,16 Moreover, a significant
number of coumarin compounds are being actively developed as
medicinal candidates or drugs with strong pharmacological activ-
ity, low toxicity and fewer side-effects, lower drug resistance, high
bioavailability, broad spectrum and better curative effects, etc.17
Several efforts have been made mainly in developing coumarin-
based anticoagulant, antioxidant, antimicrobial (antiviral, anti-
fungal and antiparasitic), anticancer, antidiabetic, analgesic, anti-
neurodegenerative and anti-inflammatory agents.18–23 Nowadays,
some coumarins proved to be enzymatic inhibitory agents [MAO
inhibitors, acetylcholinesterase (AChE) inhibitors and butyrylcho-
linesterase (BuChE) inhibitors] with potential in ND.24–27 These
studies represent an important tendency in the coumarin’s chem-
istry and biological evaluation.28,29
Early literature reported that electron delocalization and polar-
izability as well as hydrophobicity were crucial physical factors for
MAO inhibitors.30 The fused coumarins with large conjugated sys-
tem demonstrated better MAO-B inhibitory activity than the refer-
ence compounds. Therefore, fused coumarins are also worthy of
further investigation as potential MAO-B inhibitors.31 Some inter-
esting structure-activity relationships were concluded from these
studies. Literature has also reported that some compounds with
acyl or benzyloxy substituted group linked to C-3 or C-7 position
at the coumarin ring possess the ability to improve inhibitory
activity and selectivity towards MAO-B.32
In addition, previous work has been done by our group modu-
lating the positions 3 and 6 of the coumarin ring with different
substituents.33,34 Our research group has reported, in several stud-
ies, the importance of the MAO inhibitory activity of different sub-
stituents on the phenyl ring at position 3 of the 3-arylcoumarins in
the PD.35–37 Comparative studies suggested that substitutions
(methoxy and methyl groups, and bromine atoms) at meta and
para positions of the 3-aryl group played an important role in
exerting the biological activity and selectivity against MAO-B. Sev-
eral compounds proved to be better than the reference compounds
selegiline and rasagiline. Other substitutions on this moiety that
can modulate also the activity and selectivity against MAO
enzymes were also studied by our group.38 Different substitutions
of the coumarin nucleus at 3-position result in different activity
457.17 38.62
the control group of 185.67 12.06 (nmol p-tyramine/min)/mg
protein, whereas hMAO-B showed a Km of 220.33 32.80 M and
lM and a maximum reaction velocity (Vmax) in
l
Vmax of 24.32 1.97 (nmol p-tyramine/min)/mg protein (n = 5).
Most tested compounds concentration-dependently inhibited this
enzymatic control activity (Table 1).
A basic condition of any compound to act on neurodegenerative
processes is to penetrate into the brain, that is, to be able to cross
the blood–brain barrier (BBB). To examine the capability of our
compounds to pass this barrier, we selected compound 3, the most
active, with a nitro group in the phenyl at 3-position, and used a
parallel artificial membrane model.27 This is a fairly easy and suc-
cessful method to predict the passive central nervous system (CNS)
permeation, which had been previously optimized in order to be
applied to investigational compounds with limited aqueous solu-
bility.45–47 The permeability of compounds 3 through a lipid
extract of porcine brain were determined using a mixture 70:30
of phosphate buffered saline solution and ethanol (PBS/EtOH). In
each experiment 10 commercial drugs were also evaluated for
assay validation. The graphic representation of experimental per-
meability versus reported values of such well-known drugs gave
a lineal correlation, Pe (exptl) = 0.69 Pe (bibl) + 6.61 (R2 = 0.812).
From this equation and taking into account the described
limits for BBB permeation, we established that compounds with
Scheme 1. Synthesis of 3-arylcoumarins. Reagents and conditions: (i) NaH, Ac2O, rt, 20 h; (ii) H2, Pd/C, ethanol, rt, 3 h.

644
M. J. Matos et al. / Bioorg. Med. Chem. Lett. 25 (2015) 642–648
Table 1
hMAO-A and hMAO-B in vitro inhibitory activity of the synthesized derivatives 1–6 and reference compoundsa
R
H3C
O
O
Compd
R
IC50 hMAO-A (nM)
IC50 hMAO-B (nM)
S.I.b
*
1
2
3
4
5
6
20-NO2
30-NO2
40-NO2
20-NH2
30-NH2
40-NH2
—
5.52 ꢂ 103 0.30 ꢂ 103
>18d
*
6.00 0.40
>16,667d
2090
4.18 ꢂ 103 0.28 ꢂ 103c
2.10 0.10
*
2.17 ꢂ 103 0.15 ꢂ 103
>46d
>50,000d
>25,000d
3431
*
*
2.20 0.13
4.10 0.27
Selegiline
Moclobemide
67.25 ꢂ 103 1.02 ꢂ 103c
19.60 0.86
*
—
361.38 19.30
<0.0036d
a
b
c
Each IC50 value is the mean SEM from five experiments (n = 5).
Selectivity index: MAO-B selectivity ratios [IC50 (MAO-A)]/[IC50 (MAO-B)] for inhibitory effects of both new compounds and reference inhibitors.
p <0.01 regarding the corresponding IC50 obtained against MAO-B as determined by ANOVA/Dunnett’s.
d
*
Values obtained under the assumption that the corresponding IC50 against either MAO-A or MAO-B is greater than 100
Inactive at 100 M (highest concentration tested).
lM.
l
Table 2
Structural properties of the 3-aryl-6-methylcoumarin derivatives 1–6, and the reference compounds.
Compd
LogPa
MWb (Da)
TPSAc (Å2)
nOHd
nOHNHd
Volumee (Å3)
1
2
3
4
5
6
4.07
4.10
4.12
3.60
3.21
3.24
2.64
1.68
281.27
281.27
281.27
251.28
251.28
251.28
187.29
268.74
76.04
76.04
76.04
56.23
56.23
56.23
3.24
5
5
5
3
3
3
1
4
0
0
0
2
2
2
0
1
239.89
239.89
239.89
227.84
227.84
227.84
202.64
240.70
Selegiline
Moclobemide
41.57
a
b
c
LogP—expressed as the logarithm of octanol/water partition coefficient.
MW—molecular weight.
TPSA—topological polar surface area.
Number of hydrogen bond acceptors (nOH) and donors (nOHNH).
Molecular volume.
d
e
permeability values above 9.36ꢀ10ꢁ6 cm sꢁ1 could penetrate into
the CNS by passive diffusion (CNS+), whereas products with Pe
below 7.98ꢀ10ꢁ6 cm sꢁ1 could not enter (CNSꢁ). Between these
values, the CNS permeation was considered uncertain (CNS+/ꢁ).
So, compound 3 (Pe = 10.5ꢀ10ꢁ6 cm sꢁ1) would be able to cross
BBB and reach its therapeutic targets.48
Table 3
Reversibility results of hMAO-B inhibition by derivative 3 and reference inhibitors
Compd
Slope (AUF/t)a (%)
3 (2.5 nM)
Selegiline (20 nM)
Isatin (35 lM)
79.98 5.33
8.51 0.60
64.71 4.32
In order to better understand the overall properties and the
drug-like characteristics of compounds 1–6, we carried out the cal-
culation with Molinspiration software of the lipophilicity
(expressed as the octanol/water partition coefficient and herein
called logP), and the theoretical prediction of other ADME proper-
ties (molecular weight, TPSA, number of hydrogen donors and
acceptors, and volume) (see Table 2).49–51
Reversibility experiments were performed to evaluate the type
of inhibition of derivative 3, the most active compound in the ser-
ies (Table 3). An effective dilution method was used,52,53 and seleg-
iline (irreversible inhibitor) and isatin (reversible inhibitor) were
taken are standards.54 Reversibility of the inhibition shown by
compound 3 is even higher than that shown for isatin.
We also performed molecular docking simulations in the
hMAO-B to detect the hypothetical binding mode for the 3-aryl-
coumarin derivatives 1-6 and explain their structure–activity rela-
tionship.55–57 We used a similar protocol to previous studies.58 We
docked the compounds to the crystallized hMAO-B protein struc-
ture (PDB:2V60)57 using Glide SP (Standard Precision mode).56 Five
poses for each compound were extracted from the docking. The
a
Percentage values represent the mean SEM of three experiments (n = 3) rel-
ative to control; data show recovery of hMAO-B activity after dilution.
best pose was optimized using Prime MM-GBSA56 along with the
protein pocket (see Methods for a detailed description). To validate
our protocol we calculated the root mean square deviation (RMSD)
between the co-crystallized and the theoretical poses for the cou-
marins in 2V60 and 2V61.57 RMSD values were 0.82 and 1.15,
respectively.
As previous results for coumarin derivatives, the poses deter-
mined through molecular docking for the most of the studied com-
pounds placed the benzopyrone ring with the carbonyl group
oriented towards the bottom of the cavity. The methyl substituent
at position 6 is oriented towards the FAD cofactor whereas the 3-
aryl ring is placed in the hydrophobic entrance cavity. Compounds
also established a hydrogen bond between the carbonyl oxygen
and the residue Cys172. This described binding mode was found
for compounds 2–6, showing the preference for this type of mole-
cules to adopt the described conformation when interacting with

M. J. Matos et al. / Bioorg. Med. Chem. Lett. 25 (2015) 642–648
645
the protein. However, docking for compound 1 (ortho-nitro) did
not yield any poses as the one described. Figures 1 and 2 showed
the proposed binding mode extracted from the docking calcula-
tions for compounds 1–6. The different binding mode proposed
for the derivative 1 could be highly responsible for the loss of
hMAO-B activity. Leu171 showed lower van der Waals interactions
with compound 1, compared to compounds 2 and 3 with meta and
para substituents (Fig. 3a—Supplementary material). Residue
Coulomb contributions also showed a different profile between
compound 1 and compounds 2 and 3.
For instance, Phe168 and Tyr435 exhibited poor Coulomb
interaction energies with compound 1 whereas the residues
Glu84 and Pro102 showed higher Coulomb interaction energies
(Fig. 3b—Supplementary material).
Figure 2 shows the theoretical binding modes determined by
compounds 4–6 along with the important residues interacting
with the ligands. As an example, compound 5 establishes van der
Waals interactions mainly with the residues Tyr326, Ile199,
Leu171, Gln206, Cys172, Ile198, Phe168 and Ile316. Analysis of
the binding mode showed important Coulomb contributions for
residues Tyr326, Glu84 and Ile199. Compound 5 also showed a
hydrogen bond between the carbonyl oxygen and the residue
Cys172. Ligand conformation is also stabilized through different
H-arene interactions between the ligand and the residues
Leu171, Ile199 and Tyr326. However, in the comparison between
compounds 4–6, compound 4 (ortho-amino) is slightly shifted
towards the FAD cofactor (Fig. 2d). Although the ortho-amino could
establish a hydrogen bond with Tyr326, the pose determined for
compound 4 would cause the disruption of some H-arene interac-
tions, such as the interaction between the phenyl ring inTyr326
and the hydrogen at position 4 in the coumarin ring.
Moreover, compounds 4–6 had some differences in the van der
Waals and Coulomb energetic protein residue contributions.
Compound 4 (ortho-amino) showed a drastic loss of van der Waals
energy contribution with the residue Tyr326, compared to
compounds 5 and 6 with meta and para amino substituents
(Fig. 4a—Supplementary material). Moreover, lower van der Waals
contributions were captured in residues Leu171 and Cys172 for
compound 4. Coulomb interactions were also decreased for com-
pound 4 in residues Glu84 and Cys172. However, Gln206 and
Tyr326 exhibited more important electrostatic contributions in
the interaction between compound 4 and the hMAO-B protein
(Fig. 4b—Supplementary material). The amino substituent at ortho
position could cause a small displacement of the coumarin nucleus
with the consequent disruption of some stabilizer interactions
with the protein and decreasing hMAO-B activity.
(a)
Tyr326
Gln206
As represented in Table 1, most of the studied compounds
showed a selective inhibitory activity against MAO-B in the nano-
molar or micromolar range. The amino derivatives (4–6) proved to
be slightly more selective against MAO-B than the nitro derivatives
(1–3). The lowest IC50 values were obtained for compounds 2, 3, 5
and 6 (meta and para derivative compounds), being the IC50 values
in the low nanomolar range. In particular, compound 5 proved to
be more than 9-fold more active and more than 12-fold more
selective than selegiline, used as reference compound. Compounds
1 and 4, with IC50 values on the micromolar range, present on their
structure a single substituent (NO2 or NH2, respectively) in ortho
position of the phenyl at position 3. These results are consistent
with those previously described for other 6-substituted-3-
phenylcoumarins for which it was observed that the presence of
substituents in ortho position produced a decrease in the MAO-B
inhibitory activity (36). Compound 3 was the only exception
observed for this series, presenting activity against the MAO-A
FAD
Ile199
Cys172
Tyr326
Phe168
Ile316
Tyr398
(b)
(c)
Gln206
FAD
Ile199
Ile198
isoenzyme (IC50 MAO-A = 4.18 lM).
Leu171
From the data presented in Table 2, it is significant that almost
all the derivatives described possess logP values compatible with
those required to cross membranes. TPSA, described to be a predic-
tive indicator of membrane penetration, was also found to be posi-
tive. In addition, it can be observed that no violations of Lipinski’s
rule (molecular weight, logP, number of hydrogen donors and
acceptors) were found. As MAO inhibitors have to pass different
membranes and reach the central nervous system, this information
supports the potential of these derivatives. In addition, parallel
artificial membrane permeation (PAMPA) assays were performed
for compound 3, the most active derivative of the series. This com-
pound proved to cross the BBB.
Phe168
Tyr398
Cys172
Tyr326
Ile316
Gln206
FAD
Ile199
Compounds with meta and para substituents were well accom-
modated inside the hMAO-B protein pocket. The studied series
showed a hypothetical binding mode that orientates the coumarin
ring toward the FAD cofactor and the 3-aryl ring toward the
hydrophobic entrance cavity. Carbonyl in the coumarin ring is
placed in the bottom of the pocket and showed good availability
to establish a hydrogen bond with the residue Cys172. However,
the compounds with ortho substituents showed some differences
according to docking simulations. Compound 1 did not yield any
Ile198
Phe168
Tyr398
Cys172
Figure 1. Hypothetical binding mode extracted from docking calculations for
compound 1 (a), 2 (b) and 3(c). Hydrogen bonds between the compounds and the
residue Cys172 are highlighted in yellow. Main residues interacting with the
ligands are represented in tube.

646
M. J. Matos et al. / Bioorg. Med. Chem. Lett. 25 (2015) 642–648
Figure 2. Hypothetical binding mode calculated with molecular docking for compound 4 (a), 5 (b) and 6 (c) along with the main residues (tube) that interact with the
respective ligands. Hydrogen bonds with the residue Cys172 are highlighted in yellow. Compound 4 (a) also showed a hydrogen bond with the residue Tyr326. Panel (d)
shows the superposition of compounds 4 (pink color) and 5 (turquoise) inside the hMAO-B pocket. Compound 4 is slightly shifted towards the FAD cofactor.
poses as the previously described showing a completely different
binding mode that could condition the interaction with the
hMAO-B. Moreover, although the pose shown by compound 4 is
similar to the respective poses in compounds 5 and 6, the ortho-
amino substituent established a hydrogen bond with the residue
Tyr326 but at the same time led to a displacement of the coumarin
nucleus towards the FAD and caused the disruption of some
stabilizing interactions with Tyr326, Leu171 and Cys172. Binding
conformations described in this article are in agreement with
previous studies.36,37,59,60
Xunta de Galicia (Spain). M.I.R.-F. thanks the Spanish Ministry of
Economy and Competitiveness for financial support (SAF2012-
31035).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.12.
001.
Most of the studied compounds presenting structure of 3-aryl-
coumarin 1–6 behaved as potent and selective MAO-B inhibitors
in vitro. These studies showed that para and meta derivatives of
both series inhibited the activity of hMAO-B in the low nanomolar
range, while ortho derivatives inhibited the activity of the same
enzyme in the micromolar range. Compound 3 and 5 (IC50 = 2.1
and 2.2 nM, respectively) proved to be the most actives of the ser-
ies, being 5 the most selective one (9-fold more active and 12-fold
more selective than selegiline, the reference compound). In gen-
eral, the amino derivatives proved to be more selective against
MAO-B than the nitro derivatives. Molecular docking studies were
performed to establish the nature of the interaction between the
studied compounds and hMAO enzymes, leading to a rationaliza-
tion of the experimental results for the synthesized series. Addi-
tionally, prediction of ADME-related physicochemical/structural
parameters provided a preliminary indication of the great potential
of this type of compounds to cross membranes and acting in the
CNS. PAMPA assay of one of the best compounds experimentally
corroborated these results. The global results encourage us to fur-
ther explore the potential of this family of derivatives as potential
lead candidates for the treatment of PD.
References and notes
1. Trippier, P. C.; Labby, K. J.; Hawker, D. D.; Mataka, J. J.; Silverman, R. B. J. Med.
Chem. 2013, 56, 3121.
2. Meredith, G. E.; Totterdell, S.; Beales, M.; Meshul, C. K. Exp. Neurol. 2009, 219,
334.
3. Zhu, Y.; Xiao, K.; Ma, L.; Xiong, B.; Fu, Y.; Yu, H.; Wang, W.; Wang, X.; Hu, D.;
Peng, H.; Li, J.; Gong, Q.; Chai, Q.; Tang, X.; Zang, H.; Li, J.; Shen, J. Bioorg. Med.
Chem. 2009, 17, 1600.
4. Youdim, M. B. H. Exp. Neurobiol. 2010, 19, 1.
5. Yahr, M. D.; Duvoisin, R. C.; Schear, M. J.; Barrett, R. E.; Hoehn, M. M. Arch.
Neurol. 1969, 21, 343.
6. Carlsson, A. J. Neural Transm. 2002, 109, 777.
7. Abel, E. Physiol. Behav. 1995, 57, 611.
8. Engberg, G.; Elebring, T.; Nissbrandt, H. J. Pharmacol. Exp. Ther. 1991, 259, 841.
9. Scarpini, E.; Scheltens, P.; Feldman, H. Lancet Neurol. 2003, 2, 539.
10. Bortolato, M.; Chen, K.; Shih, J. C. Adv. Drug Deliv. Rev. 2008, 60, 1527.
11. Nagatsu, T. Neurotoxicology 2004, 25, 11.
12. Venugopala, K. N.; Rashmi, V.; Odhav, B. BioMed. Res. Int. 2013, 2013. 963248.
13. Murray, R. D. H. Prog. Chem. Org. Nat. Prod. 2002, 83, 1.
14. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Curr. Med. Chem.
2005, 12, 887.
15. Qian, L.-L.; Han, X.-E.; Han, H.; Chen, X.-Z.; Yuan, H.-H. Guangzhou Huagong
2013, 41, 41.
16. Zheng, L.; Zhao, T.; Sun, L.-X. Shizhen Guoyi Guoyao 2013, 24, 714.
17. Borges, F.; Roleira, F.; Milhazes, N.; Uriarte, E.; Santana, L. Front. Med. Chem.
2009, 4, 23.
18. Bubols, G. B.; Vianna, D. R.; Medina-Remon, A.; von Poser, G.; Lamuela-
Raventos, R. M.; Eifler-Lima, V. L.; Garcia, S. C. Mini Rev. Med. Chem. 2013, 13,
318.
Acknowledgments
19. Xia, L.-X.; Wang, Y.-B.; Huang, W.-L.; Qian, H. Zhongguo Xinyao Zazhi 2013, 22,
2392.
20. Kapoor, S. Cytotechnology 2013, 65, 787.
21. Bansal, Y.; Sethi, P.; Bansal, G. Med. Chem. Res. 2013, 22, 3049.
22. O’Kennedy, R. Biology, Applications and Mode of Action In Thornes, R. D., Ed.;
John Wiley and Sons: New York, USA, 1997.
23. Kontogiorgis, C.; Detsi, A.; Hadjipavlou-Litina, D. Exp. Opin. Therap. Patents
2012, 22, 437.
This work was partially supported by Spanish Researchers
Personal Funds and University of Santiago de Compostela. M.J.M.
thanks Fundação para a Ciência e Tecnologia for her postdoctoral
grant (SFRH/BPD/95345/2013). S.V. thanks the Plan Galego de
Investigación, Innovación
e
Crecemento 2011–2015 (I2C),
European Social Fund (ESF) and Angeles Alvariño Program from

M. J. Matos et al. / Bioorg. Med. Chem. Lett. 25 (2015) 642–648
647
24. Patil, P. O.; Bari, S. B.; Firke, S. D.; Deshmukh, P. K.; Donda, S. T.; Patil, D. A.
Bioorg. Med. Chem. 2013, 21, 2434.
25. Anand, P.; Singh, B. Arch. Pharmacal Res. 2013, 36, 375.
26. Huang, L.; Su, T.; Li, X. Curr. Top. Med. Chem. 2013, 13, 1864.
27. Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. Eur. J. Med. Chem. 2003,
38, 223.
28. Anand, P.; Singh, B.; Singh, N. Bioorg. Med. Chem. 2012, 20, 1175.
29. Helguera, A. M.; Perez-Machado, G.; Cordeiro, M. N. D. S.; Borges, F. Mini Rev.
Med. Chem. 2012, 12, 907.
30. Pisani, L.; Muncipinto, G.; Miscioscia, T. F.; Nicolotti, O.; Leonetti, F.; Catto, M.;
Caccia, C.; Salvati, P.; Soto-Otero, R.; Mendez-Alvarez, E.; Passeleu, C.; Carotti,
A. J. Med. Chem. 2009, 52, 6685.
31. Secci, D.; Carradori, S.; Bolasco, A.; Chimenti, P.; Yáñez, M.; Ortuso, F.; Alcaro, S.
Eur. J. Med. Chem. 2011, 46, 4846.
32. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Bizzarri, B.; Granese, A.;
Carradori, S.; Yáñez, M.; Orallo, F.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2009, 52,
1935.
33. Matos, M. J.; Viña, D.; Vazquez-Rodriguez, S.; Uriarte, E.; Santana, L. Curr. Top.
Med. Chem. 2012, 12, 2210.
The previously prepared 6-methyl-3-nitrophenylcoumarin (1,
2
or 3,
2.46 mmol) was dissolved in ethanol (5 mL) and a catalytic amount of Pd/C
was added to the mixture. The solution was stirred, at room temperature,
under a H2 atmosphere, for 3 h. After completion of the reaction, the mixture
was filtered to eliminate the catalyst. The obtained crude was then purified by
flash chromatography (hexane/ethyl acetate 9:1) to give the desired coumarin
4–6 as white solids in yields between 92% and 97%.
3-(20-Aminophenyl)-6-methyl-coumarin (4): Yield: 92%. Mp: 139–140 °C.40
3-(30-Aminophenyl)-6-methyl-coumarin (5): Yield: 95%. Mp: 156–157 °C. 1H
NMR (300 MHz; CDCl3): d = 2.48 (s, 3H, CH3), 3.98 (s, 2H, NH2), 7.32–7.35 (m,
1H, H-40), 7.32–7.40 (m, 2H, H-50, H-60), 7.55–7.58 (m, 1H, H-7), 7.69–7.72 (m,
1H, H-8), 7.82 (s, 1H, H-20), 7.87–7.91 (m, 1H, H-5), 7.92 (s, 1H, H-4). 13C NMR
(75 MHz; CDCl3): d = 24.9, 116.3, 125.4, 127.2, 127.8, 129.9, 130.0, 130.1, 131.4,
131.7, 132.9, 134.2, 134.4, 140.5, 151.8, 191.9. DEPT (75 MHz; CDCl3): d = 24.9,
116.3, 127.2, 127.8, 129.9, 131.4, 131.7, 132.9, 140.5. MS (EI, 70 eV): m/z (%):
252 (18), 251 (M+, 90), 236 (90), 178 (18), 154 (19), 147 (21), 134 (58), 129
(25), 125 (18), 116 (20), 112 (37), 111 (29), 109 (17). Anal. Calcd for C16H13NO2:
C, 76.48; H, 5.21. Found: C, 76.39; H, 5.15.
3-(40-Aminophenyl)-6-methyl-coumarin (6): Yield: 97%. Mp: 191–192 °C.41
43. Pharmacological assays—General methods: The tested compounds were
dissolved in DMSO (Sigma–Aldrich, Alcobendas, Madrid, Spain) to prepare
10 mM stock solutions which were kept for storage at ꢁ20 °C. Percentage of
DMSO used in the experiments was never higher than 1%. Selegiline, used as
reference inhibitor, have been acquired from Sigma-Aldrich (Alcobendas,
Madrid, Spain). Moclobemide has been kindly provided by Hoffman-La Roche
Laboratories (Basel, Switzerland). Human recombinant MAO isoforms, used in
the experiments, was purchased from Sigma-Aldrich (Alcobendas, Madrid,
Spain). Resorufin sodium salt, p-tyramine hydrochloride, sodium phosphate
buffer, horseradish peroxidase and AmplexÒ Red reagent has been supplied in
the assay kit of AmplexÒ Red MAO Molecular Probes (Molecular Probes, Inc.,
Eugene, Oregon, USA).
34. Santana, L.; González-Díaz, H.; Quezada, E.; Uriarte, E.; Yáñez, M.; Viña, D.;
Orallo, F. J. Med. Chem. 2008, 51, 6740.
35. Matos, M. J.; Vilar, S.; González-Franco, R. M.; Uriarte, E.; Santana, L.; Friedman,
C.; Tatonetti, N. P.; Viña, D.; Fontenla, J. A. Eur. J. Med. Chem. 2013, 63, 151.
36. Ferino, G.; Cadoni, E.; Matos, M. J.; Quezada, E.; Uriarte, E.; Santana, L.; Vilar, S.;
Tatonetti, N. P.; Yañez, M.; Viña, D.; Picciau, C.; Serra, S.; Delogu, G. Chem. Med.
Chem. 2013, 8, 956.
37. Matos, M. J.; Terán, C.; Pérez-Castillo, Y.; Uriarte, E.; Santana, L.; Viña, D. J. Med.
Chem. 2011, 54, 7127.
38. Viña, D.; Matos, M. J.; Ferino, G.; Cadoni, E.; Laguna, R.; Borges, F.; Uriarte, E.;
Santana, L. Chem. Med. Chem. 2012, 7, 464.
39. Delogu, G.; Picciau, C.; Ferino, G.; Quezada, E.; Podda, G.; Uriarte, E.; Viña, D.
Eur. J. Med. Chem. 2011, 46, 1147.
40. Matos, M. J.; Gaspar, A.; Borges, F.; Uriarte, E.; Santana, L. Org. Prep. Proc. Int.
2012, 44, 522.
41. Rao, D. V.; Sayigh, A. A. R.; Ulrich H. US 3644413 A 19720222, Chem. Abstr., 77,
7318, 1972.
42. Chemistry—General methods: Starting materials and reagents were obtained
from commercial suppliers and were used without further purification (Sigma–
Aldrich). Melting points (mp) are uncorrected and were determined with a
Reichert Kofler thermopan or in capillary tubes in a Büchi 510 apparatus. 1H
NMR (300 MHz) and 13C NMR (75.4 MHz) spectra were recorded with a Bruker
AMX spectrometer using DMSO-d6 or CDCl3 as solvent. Chemical shifts (d) are
expressed in ppm using TMS as an internal standard. Coupling constants (J) are
expressed in Hz. Spin multiplicities are given as s (singlet), d (doublet), and m
(multiplet). Mass spectrometry was carried out with a Hewlett-Packard-5972-
MSD spectrometer. Elemental analyses were performed with a PerkinElmer
240B microanalyzer and are within 0.4% of calculated values in all cases. Flash
chromatography (FC) was performed on silica gel (Merck 60, 230–400 mesh);
analytical TLC was performed on pre-coated silica gel plates (Merck 60 F254).
Organic solutions were dried over anhydrous Na2SO4. Concentration and
evaporation of the solvent after reaction or extraction was carried out on a
rotary evaporator (Büchi Rotavapor) operating at reduced pressure. The
analytical results showed >95% purity for all compounds.
Determination of MAO isoforms enzymatic activity: Briefly, 0.1 mL of sodium
phosphate buffer (0.05 M, pH 7.4) containing different concentrations of the
test drugs (new compounds or reference inhibitors) in various concentrations
and adequate amounts of recombinant hMAO-A or hMAO-B required and
adjusted to obtain in our experimental conditions the same reaction velocity,
that is, to oxidize (in the control group) the same concentration of substrate:
165 pmol of p-tyramine/min (hMAO-A: 1.1
150 nmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg
protein; hMAO-B: 7.5 g protein; specific activity: 22 nmol of p-tyramine
lg protein; specific activity:
l
transformed/min/mg protein) were incubated for 15 min at 37 °C in a flat-
black-bottom 96-well microtest plate, placed in the dark fluorimeter chamber.
After this incubation period, the reaction was started by adding (final
concentrations) 200
peroxidase and 1 mM p-tyramine. The production of H2O2 and, consequently,
of resorufin was quantified at 37 °C in multidetection microplate
lM
AmplexÒ Red reagent, 1 U/mL horseradish
a
fluorescence reader (FLX800, Bio-Tek Instruments, Inc., Winooski, VT, USA)
based on the fluorescence generated (excitation, 545 nm, emission, 590 nm)
over a 15 min period, in which the fluorescence increased linearly.43 Control
experiments were carried out simultaneously by replacing the test drugs (new
compounds and reference inhibitors) with appropriate dilutions of the
vehicles. In addition, the possible capacity of the above test drugs to modify
the fluorescence generated in the reaction mixture due to non-enzymatic
inhibition (e.g., for directly reacting with AmplexÒ Red reagent) was deter-
mined by adding these drugs to solutions containing only the AmplexÒ Red
reagent in a sodium phosphate buffer. To determine the kinetic parameters of
hMAO-A and hMAO-B (Km and Vmax), the corresponding enzymatic activity of
both isoforms was evaluated (under the experimental conditions described
above) in the presence of a number (a wide range) of p-tyramine concentra-
tions. The specific fluorescence emission (used to obtain the final results) was
calculated after subtraction of the background activity, which was determined
from wells containing all components except the hMAO isoforms, which were
replaced by a sodium phosphate buffer solution. In our experimental condi-
tions, this background activity was practically negligible. MAO activity of the
test compounds and reference inhibitors is expressed as IC50, ie the concen-
tration of each drug required to produce a 50% decreased on control value
activity isoforms MAO.
General procedure for the synthesis of 6-methyl-3-nitrophenylcoumarins 1–3: To a
dry 100-mL round-bottomed flask, the ortho-hydroxy-5-methylbenzaldehyde
(42.5 mmol), the conveniently substituted nitrophenylacetic acid (42.5 mmol)
and acetic anhydride (40.1 mL, 0.43 mol) were added. Then, sodium hydride
(60% dispersion in mineral oil, 42.5 mmol) was added in small aliquots. After
the dissolution of the reagents, precipitation process was observed (2–5 min).
The reaction mixture was stirred for 20 h, and then water (7 mL) was added.
After the addition of acetic acid (43 mL), the mixture was cooled to 4 °C for 4 h.
The resulting precipitate was filtered and washed with cold glacial acetic acid.
The acetic acid was then removed as an azeotrope upon addition of 250 mL
toluene and evaporated to dryness. The process was repeated three times. The
final residue was dried under vacuum and purified by FC (hexane/ethyl acetate
9:1) to give the products 1–3 as beige powders, in yields between 70–80%.
6-Methyl-3-(20-nitrophenyl)coumarin (1): Yield: 70%. Mp: 95–96 °C. 1H NMR
(300 MHz; DMSO-d6): d = 2.39 (s, 3H, CH3), 7.21–7.23 (m, 1H, H-40), 7.31–7.37
(m, 2H, H-7, H-8), 7.39–7.48 (m, 3H, H-5, H-50, H-60), 7.63 (d, J = 2.3 Hz, 1H, H-
30), 7.75 (s, 1H, H-4). 13C RMN (75 MHz; DMSO-d6): d = 20.8, 116.4, 118.7,
123.7, 127.4, 127.8, 128.6, 130.1, 131.4, 132.9, 133.0, 134.2, 135.7, 142.5, 152.2,
160.9. DEPT (75 MHz; DMSO-d6): d = 20.8, 116.4, 127.4, 127.8, 130.1, 131.4,
132.9, 133.0, 142.5. MS (EI, 70 eV): m/z (%): 282 (14), 281 (M+, 72), 178 (14),
136 (23), 90 (18). Anal. Calcd for C16H11NO4: C, 68.32; H, 3.94. Found: C, 68.33;
H, 3.96.
44. Yáñez, M.; Fraiz, N.; Cano, E.; Orallo, F. Biochem. Biophys. Res. Commun. 2006,
344, 688.
45. Rodríguez-Franco, M. I.; Fernández-Bachiller, M. I.; Pérez, C.; Hernández-
Ledesma, B.; Bartolomé, B. J. Med. Chem. 2006, 49, 459.
46. Fernández-Bachiller, M. I.; Pérez, C.; Monjas, L.; Rademann, J.; Rodríguez-
Franco, M. I. J. Med. Chem. 2012, 55, 1303.
47. López-Iglesias, B.; Pérez, C.; Morales-García, J. A.; Alonso-Gil, S.; Pérez-Castillo,
A.; Romero, A.; López, M. G.; Villarroya, M.; Conde, S.; Rodríguez-Franco, M. I. J.
Med. Chem. 2014, 57, 3773.
6-Methyl-3-(30-nitrophenyl)coumarin (2): Yield: 74%. Mp: 96–97 °C. 1H NMR
(300 MHz; DMSO-d6): d = 2.37 (s, 3H, CH3), 7.21 (d, J = 8.2 Hz, 2H, H-7, H-8),
7.50–7.56 (m, 2H, H-5, H-50), 7.87 (d, J = 6.5 Hz, 1H, H-60), 8.17 (s, 1H, H-4),
8.28–8.37 (m, 2H, H-20, H-40). 13C NMR (75 MHz; DMSO-d6): d = 20.6, 122.6,
123.9, 125.1, 127.9, 130.2, 131.4, 136.4, 136.6, 136.8, 137.2, 148.1, 149.3, 170.2,
190.6. DEPT (75 MHz; DMSO-d6): d = 20.6, 122.6, 123.9, 125.1, 130.2, 131.4,
136.6, 137.2, 190.6. MS (EI, 70 eV): m/z (%): 282 (8), 281 (M+, 43), 253 (7), 235
(8), 178 (15), 163 (10), 136 (38), 90 (15). Anal. Elem. Calcd for C16H11NO4: C,
68.32; H, 3.94. Found: C, 68.38; H, 3.99.
48. In vitro blood-brain barrier permeation assay: Prediction of the brain penetration
was evaluated using a PAMPA-BBB assay, in a similar manner as previously
described.27,45–47 Pipetting was performed with
a semi-automatic robot
(CyBiÒ-SELMA) and UV reading with a microplate spectrophotometer (Multis-
kan Spectrum, Thermo Electron Co.). Commercial drugs, phosphate buffered
saline solution at pH 7.4 (PBS), and dodecane were purchased from Sigma,
Aldrich, Acros, and Fluka. Millex filter units (PVDF membrane, diameter
25 mm, pore size 0.45
lipid (PBL) was obtained from Avanti Polar Lipids. The donor microplate was a
96-well filter plate (PVDF membrane, pore size 0.45 m) and the acceptor
lm) were acquired from Millipore. The porcine brain
6-Methyl-3-(40-nitrophenyl)coumarin (3): Yield: 80%. Mp: 100–101 °C.20
General procedure for the synthesis of 3-aminophenyl-6-methylcoumarins 4–6:
l

648
M. J. Matos et al. / Bioorg. Med. Chem. Lett. 25 (2015) 642–648
microplate was an indented 96-well plate, both from Millipore. The acceptor
96 well microplate was filled with 200 L of PBS/ethanol (70:30) and the filter
the reaction was monitored for 15 min. Reversible inhibitors show linear
l
progress with a slope equal to ꢃ91% of the slope of the control sample, whereas
irreversible inhibition reaches only ꢃ9% of this slope. Control tests were
carried out by pre-incubating and diluting the enzyme in the absence of
inhibitor.
surface of tꢁh1e donor microplate was impregnated with 4 mL of PBL in dodecane
(20 mg mL ). Compounds were dissolved in PBS/ethanol (70:30) at
100
wells (200
l
g mLꢁ1, filtered through a Millex filter, and then added to the donor
lL). The donor filter plate was carefully put on the acceptor plate to
54. Gerlach, M.; Riederer, P.; Youdim, M. B. Eur. J. Pharmacol. 1992, 226, 97.
55. Molecular docking calculations: We performed molecular docking simulations
using the Schrödinger package.56
form a sandwich, which was left undisturbed for 240 min at 25 °C. After
incubation, the donor plate is carefully removed and the concentration of
compounds in the acceptor wells was determined by UV–vis spectroscopy.
Every sample is analyzed at five wavelengths, in four wells and at least in three
independent runs, and the results are given as the mean standard deviation.
In each experiment, 10 quality control standards of known BBB permeability
were included to validate the analysis set.
Ligand preparation: Our dataset is made out of the coumarin derivatives 1–6
and the co-crystallized coumarins in 2V60 and 2V61 (PDB code)57 used to
validate the docking protocol. We used LigPrep module56 to generate possible
protonation states at pH 7.0 2.0, possible tautomers, and optimized struc-
tures for all the ligands.
49. Theoretical evaluation of absorption, distribution, metabolism and excretion
properties: The absorption, distribution, metabolism and excretion (ADME)
properties of the studied compounds were calculated using the Molinspiration
property programme. LogP was calculated using the methodology developed
by Molinspiration as a sum of fragment-based contributions and correction
factors (48). Topological polar surface area (TPSA) was calculated based on the
methodology published by Ertl et al. as a sum of fragment contributions.
Oxygen and nitrogen-centred polar fragments were considered (48). Polar
surface area (PSA) has been shown to be a very good descriptor characterizing
drug absorption, including intestinal absorption, bioavailability, Caco-2
permeability and blood–brain barrier penetration. The method for
calculation of molecule volume developed at Molinspiration is based on
group contributions. These have been obtained by fitting the sum of fragment
contributions to ‘real’ threedimensional (3D) volume for a training set of about
12,000, mostly drug-like molecules. 3D molecular geometries for a training set
were fully optimized by the semi-empirical AM1 method.
50. M cheminformatics, Bratislava, Slovak Republic, http://www.molinspiration.
com/services/properties.html.
51. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
1997, 23, 3.
52. Copeland, R. A. Evaluation of Enzyme Inhibitors in Drug Discovery; Wiley-
Interscience: Hoboken, 2005.
53. Determination of inhibition mode: To evaluate whether compounds 8, 14, and 15
are reversible or irreversible hMAO-B inhibitors, a dilution method was used.52
A 100ꢂ concentration of the enzyme used in the above described experiments
was incubated with a concentration of inhibitor equivalent to 10-fold its IC50
value. After 30 min, the mixture was diluted 100-fold into reaction buffer
containing Amplex Red reagent, horseradish peroxidase, and p-tyramine, and
Protein preparation: We used the crystal structure of the hMAO-B (PDB:2V60)
to run the docking simulations. In 2V60 the protein is bound to the coumarin
derivative c17. We preprocessed the protein structure with the Protein
Preparation Wizard.56 In this step we assigned bond orders, added hydrogens,
created disulfide bonds, filled possible missing side chains, added cap termini,
deleted water molecules except the water that establishes a hydrogen bond
with the co-crystallized coumarin derivative c17 and optimized the H-bond
network, including reorientation of hydroxyl groups and optimization of the
protonation state of some residues.
Receptor grid: As a previous step to docking, we generated a receptor grid
centered in the coumarin derivative c17 with a length of 20 Å. The van der
Waals scaling factor was 1.0 with 0.25 as a partial charge cut-off.
Molecular docking procedure: We performed molecular docking simulations in
the hMAO-B using Glide Standard Precision level.56 For each ligand, five poses
were retained. The final pose selection was made based on the energy
score Emodel. Protein pocket and ligand poses were optimized using Prime
MM-GBSA.56
56. Schrödinger Package, Schrödinger, LLC, New York, NY, 2014. https://
www.schrodinger.com.
57. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.
E.; Mattevi, A. J. Med. Chem. 2007, 50, 5848.
58. Matos, M. J.; Janeiro, P.; González-Franco, R. M.; Vilar, S.; Tatonetti, N. P.;
Santana, L.; Uriarte, E.; Borges, F.; Fontenla, J. A.; Viña, D. Fut. Med. Chem. 2014,
6, 371.
59. Matos, M. J.; Vilar, S.; García-Morales, V.; Tatonetti, N. P.; Uriarte, E.; Santana,
L.; Viña, D. Chem. Med. Chem. 2014, 9, 1488.
60. Ferino, G.; Vilar, S.; Matos, M. J.; Uriarte, E.; Cadoni, E. Curr. Top. Med. Chem.
2012, 12, 2145.